Electrochemical Study of Astaxanthin and ... - ACS Publications

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Electrochemical Study of Astaxanthin and Astaxanthin n‑Octanoic Monoester and Diester: Tendency to Form Radicals A. Ligia Focsan,*,† Shanlin Pan,‡ and Lowell D. Kispert‡ †

Department of Chemistry, Valdosta State University, Valdosta, Georgia 31689, United States Department of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487-0336, United States

‡

S Supporting Information *

ABSTRACT: The carotenoid astaxanthin known for its powerful antioxidant activity was electrochemically investigated along with the synthesized astaxanthin noctanoic monoester and astaxanthin n-octanoic diester. Cyclic voltammograms (CVs) revealed a two-electron transfer oxidation for all three carotenoids with a difference in the two oxidation potentials (ΔE = E20 − E10) slightly increasing from astaxanthin to the monoester to diester. Minimal or no exposure to water prevented the formation of carotenoid neutral radicals from dications and radical cations, causing near absence of the fifth peak in the CVs. This makes the CVs almost reversible and enables a more precise simulation of the redox potentials and the equilibrium constants for the formation of radical cations. The first oxidation potential (E10) of 0.7678, 0.7738, and 0.7753 V versus SCE and the second oxidation potential (E20) of 0.9828, 0.9931, and 0.9966 V versus SCE for astaxanthin, monoester, and diester, respectively, have been standardized to the potential of ferrocene of 0.528 V vs SCE given in a previous study. Reduction potentials (E30) for formation of carotenoid neutral radicals from dications after proton loss from the three studied carotenoids are presented and compared to those of other carotenoids. According to our DFT calculations, the most favorable sites for deprotonation of radical cations and dications are found on the cyclohexene rings. These measurements provide insight into important properties of these carotenoids like radical scavenging of • OH, •CH3, and •OOH by proton abstraction from the carotenoid or the formation of carotenoid neutral radicals from radical cations which can quench photoexcited states. There is no essential difference in first oxidation potentials for the three carotenoids, which suggests a similar scavenging rate of the esters of astaxanthin toward •OH, •CH3, and •OOH radicals when compared to astaxanthin itself. The large equilibrium constants Kcom (102.4, 409.6, and 204.8 for astaxanthin, monoester, and diester) derived from simulation indicate a preference for radical cation formation for both astaxanthin and its esters, while electron transfer to form dications will be unlikely. Proton transfer from the radical cations, which are weak acids, to the neighboring proton acceptors will form neutral radicals, which allows quenching of excited states.

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from approximately 0.2 × 10−2 to 5.0 × 10−2 cm/s in 0.1 M tetrabutylammoniumhexafluorophosphate (TBAHFP) and anhydrous methylene chloride, reflecting a fast heterogeneous electrotransfer for the electrooxidation in aprotic solvents.2 The oxidation potentials for synthesized carotenoids4 fall in the same range as those for natural carotenoids. However, variations in the structure of carotenoids, like changing substituents and/or number of double bonds in the carotenoid structure, lead to variations in redox potentials. For example, the difference in oxidation potentials (ΔE = E10 − E20) depends upon the electron-withdrawing strength of the carotenoid substituents.4 With an increase in electron-withdrawing strength of substituents, the separation between the two oxidation waves increases. This also increases the comproportionation equilibrium constant (Kcom).5 In solution, the radical cation, dication, and neutral carotenoid species coexist in a

INTRODUCTION Numerous carotenoids (∼750) have now been isolated from natural sources.1 Many of them have been studied electrochemically2−4 to determine whether there is a relationship between their structure and electrochemical properties. Electrochemical properties of carotenoids help in understanding their roles in different biosystems as light harvesting agents, photoprotect devices, or antioxidants. The redox potentials and other electrochemical parameters can describe the electron transfer properties and the chemical stability and reactivity of carotenoid radicals, radical cations, and dications, as well as other radical species. Carotenoids exhibit relatively low oxidation potentials, making them good electron donors. It was reported2 that βcarotene, canthaxanthin, 8′-apo-β-carotene-8′-al, and other naturally occurring carotenoids in anhydrous methylene chloride have varying oxidation potentials versus SCE with E10 ranging from 0.53 to 0.76 V and E20 from 0.55 to 0.95 V referenced to the oxidation potential of ferrocene of 0.428 V. The electron transfer heterogeneous rate constants (ks) vary © 2014 American Chemical Society

Received: December 11, 2013 Revised: February 3, 2014 Published: February 4, 2014 2331

dx.doi.org/10.1021/jp4121436 | J. Phys. Chem. B 2014, 118, 2331−2339

The Journal of Physical Chemistry B

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Also, it was reported2 that tests were performed with other working electrodes like Pt (used in the present study) and a similar behavior was observed. Astaxanthin (in Chart 1) investigated in the current study has the most powerful antioxidant activity on singlet oxygen quenching: it is 40 times more effective as an antioxidant than β-carotene and more than 100 times more effective than vitamin E. Astaxanthin has numerous clinically proven benefits in neurovascular and cardiovascular health, skin aging defense, fertility, diabetes, eye fatigue relief, immune system booster, etc. The richest source of astaxanthin, Haematococcus pluvialis alga, contains only 5% free astaxanthin. The largest carotenoid fraction is given by monoesters of astaxanthin, about 70%. Diesters of astaxanthin constitute about 10%, and the rest of carotenoid fraction is composed of other carotenoids.6 Accumulation of astaxanthin and its mono- and diesters in high concentration in several unicellular algae is considered to be a survival strategy under photooxidative and salt stress.7,8 According to the experimental in vitro studies, astaxanthin esters function as powerful quenchers of singlet oxygen under both hydrophilic and hydrophobic conditions9 and, possibly, astaxanthin esters may have an even higher quenching ability.10 However, the detailed mechanisms of photoprotective and antioxidant functions of astaxanthin and its esters remain open to question. It was shown that the antioxidant activity of carotenoids could be related to their chemical composition. The structure of carotenoids, the number of double bonds, and the presence of functional groups may influence the scavenging properties.11,12 The scavenging reactions not only depend on the nature of carotenoids but also depend on the nature of the free radical.13 Carotenoids can react with free radicals by different pathways: radical addition to the carotenoid polyene chain, electron transfer yielding the carotenoid radical cation,13,14 or possibly hydrogen abstraction from the C4-position of the cyclohexene ring.12 For example, studies have shown that there are particular radicals that cause electron transfer and radicals that only lead to addition reactions.14 The role of environment was also suggested to be important, with the polarity of solvents leading to different mechanisms of interaction of radicals with carotenoids.14 We have shown15 earlier that the ability of carotenoids to scavenge •OH, •CH3, and •OOH radicals generated in a Fenton reaction increases significantly with increasing first oxidation potential. This study looks into the electrochemical properties of astaxanthin and its n-octanoic mono- and diesters to gain insight into their roles in Haematococcus pluvialis as photoprotect devices and/or as antioxidants. The structures and numbering system used in this paper for astaxanthin, n-octanoic acid monoester, and noctanoic acid diester are given in Chart 1.

comproportionation equilibrium and the value of the equilibrium constant depends on the nature of substituents on the terminal ends of the carotenoid. For example, in the case of β-carotene (Chart 1) with cyclohexene ends as electronChart 1. Structures of the Carotenoids β-Carotene, Astaxanthin, n-Octanoic Acid Monoester, and n-Octanoic Acid Diester and Their Numbering System

donating groups, a small comproportionation equilibrium constant2 favors the presence of the dications in solution. On the other hand, canthaxanthin with two strong electronaccepting terminal groups like carbonyl has a large comproportionation equilibrium constant2 favoring formation of radical cations in solution. This is in accordance with the reported oxidation potential inversion for β-carotene and the reduction potential inversion for canthaxanthin.2 According to the study,2 hole-accepting terminal groups for oxidations as in β-carotene localize the charges in dication, leading to potential inversion. The carotenoid β-carotene displays only a single oxidation peak for the two-electron transfer, thus making the identification of the two oxidation potentials difficult. The second oxidation potential (E20) which corresponds to oxidation of radical cations and formation of dications of βcarotene was determined to be lower than the oxidation potential of neutral carotenoid species (E10) corresponding to formation of radical cations. This potential inversion in oxidation is due to a disfavored stabilization of the radical cation by interaction with the solvent due to its charge delocalization over the entire molecular framework. Localization of charges in dication toward the ends of the molecule at large distance from one another minimizes the Coulombic repulsion and favors the solvation of the dication, providing additional stabilization. Potential inversion also occurs in the reduction of canthaxanthin, where electron-accepting terminal groups like carbonyl favor localization of charges in dianion. The precise measured values for the oxidation potentials of βcarotene and canthaxanthin by Hapiot et al.2 provided a standard reference for carotenoids which will be used in the present study. The oxidation potentials for β-carotene and canthaxanthin in methylene chloride were calibrated versus ferrocene (E10 = 0.528 V vs SCE), and the electrochemical cell used a Pt wire as the counter electrode, SCE as the reference electrode, and a Au (1 mm) disk as the working electrode.2

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METHODS AND MATERIALS Astaxanthin purchased form Sigma (99%) was stored at −14 °C in a desiccator. The sample purity was determined by 1H NMR (360 MHz, CDCl3, Cambridge Isotope Laboratories). No unaccounted NMR lines were observed, and the purity of astaxanthin was estimated to be better than 98%. Esters of astaxanthin, n-octanoic acid monoester, and n-octanoic acid diester were synthesized according to Fukami et al.16 Purification was achieved on a chromatography column using Silicycle silica gel. To confirm the structure of synthesized ester derivatives, 0.7 mg of carotenoid (astaxanthin, n-octanoic acid monoester, or n-octanoic acid diester of astaxanthin) in 0.5 mL 2332



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of CDCl3 solutions were prepared and NMR spectra were collected utilizing a Bruker spectrometer 360 MHz BrukerAvance Spectrometer Bruker/MagnexUltraShield 360 MHz magnet. Spectra were processed using the Mestrec program. Cyclic voltammetry (CV) was carried out using the Bio Analytical Systems BAS-100W electrochemical analyzer. The auxiliary electrode was a platinum wire, the working electrode was a platinum electrode with a 1.6 mm diameter purchased from BASi, and the reference electrode was a saturated calomel electrode (SCE) purchased from CH Instruments, Inc. Carotenoids’ quantities were measured in the air, placed in the volumetric flasks covered with parafilm in which a few holes were pinched, transferred to a drybox, and evacuated three times. All used glassware and CV cells (except calomel electrode) sat in the oven at 120 °C for at least 24 h before being used and transferred to the drybox in a desiccator. All carotenoid solutions were prepared in the drybox under a nitrogen atmosphere using anhydrous methylene chloride (CH2Cl2, 99.99%, Aldrich) from a Sure Seal bottle used without further purification and extracted with a glass syringe. The dichloromethane solutions contain 0.0004 M carotenoids (astaxanthin, astaxanthin n-octanoic monoester, or astaxanthin n-octanoic diester) and 0.1 M TBAHFP as the supporting electrolyte. The electrolyte supplied by Fluka was also opened and kept in the drybox under a nitrogen atmosphere. For internal standardization, a 0.001 M solution of ferrocene was added. The CV cell containing the solution, the auxiliary electrode, and the working electrode was assembled in the drybox and sealed. The saturated calomel electrode was added to the assembly outside the drybox after nitrogen gas had been passed through the solution. Nitrogen gas was flowing through the cell during the whole experiment. The working electrode was polished, and the electrodes were cleaned using acetone and rinsed with dichloromethane. DFT calculations for dications and cations of astaxanthin and β-carotene were performed with the Gaussian 09 program package17 on the Cray XD1 computer at the Alabama Supercomputer Center. Geometries were optimized at the B3LYP/6-31G** level,18,19 which we have previously shown20 to be suitable for predicting the geometry of β-carotene-based radicals.

Scheme 1. Reaction Mechanism of Carotenoids

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Figure 1. Comparison of CVs at a scan rate of 500 mV/s. Current was normalized in the cathodic direction. Ferrocene was used as an internal reference label.

RESULTS AND DISCUSSION According to Scheme 1 as given in previous studies,2,4 during an oxidation−reduction cycle, radical cations (Car•+), dications (Car2+), cations (#Car+), and neutral radicals (#Car•) are formed in solution at room temperature. During the anodic scan, two separate peaks, 1 and 2 (see Figure 1), correspond to the oxidation of neutral carotenoid species (eq 1 in Scheme 1) and, upon transfer of the second electron, to the oxidation of the radical cations (eq 2 in Scheme 1). This leads to formation of radical cations and dications, respectively, with the corresponding oxidation potentials, E10 and E20. The two cathodic peaks, 3 and 4 (see Figure 1), are due to the reduction of the dications (reversed eq 2, Scheme 1) and radical cations (reversed eq 1, Scheme 1) with formation of radical cations and neutral carotenoid species, respectively. In previous electrochemical studies,2,4 a fifth peak was present in the low potential region near 0.1−0.3 V and was attributed to the formation of neutral radicals #Car• formed by reduction of the cation #Car+ (eq 3, Scheme 1), previously formed from deprotonation of the dication Car2+ (eq 5, Scheme 1). Neutral radicals were also shown to occur upon deprotonation from the radical cations

(eq 6, Scheme 1). A reduction potential, E30, is attributed to the formation of neutral radicals. The fifth peak occurs only when the dications have been formed, and its intensity depends on the switch potentials that control the amount of dication formed. If the potential is switched before the dication can form, there is no fifth peak occurring in the CV.2 Two well-defined sets of quasi-reversible peaks indicating that radical cations and dications have a relatively long lifetime were observed for astaxanthin, n-octanoic acid monoester, and n-octanoic acid diester of astaxanthin (Figure 1). Upon simulation, which included a number of measurements at different scan rates (50, 100, 300, 500, 700, 1000, 2000, 3000, 4000, and 5000 mV/s), the first oxidation potential E10 was found to be 0.6886, 0.6946, and 0.6961 V for astaxanthin, monoester, and diester, respectively (see Table 1). The second oxidation potentials E20 were 0.9036, 0.9139, and 0.9174 V, respectively. These potentials were referenced to the potential of ferrocene of 0.4488 V versus SCE. They were further standardized to the measured potential of 0.528 V of Hapiot et 2333



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Table 1. Redox Potentials of Astaxanthin and Its Esters; V vs SCE compound ferrocene astaxanthin monoester astaxanthin diester astaxanthin

E10

E20

E30

E10 corrected to 0.528 V



ΔE

Kcom from simulation

0.4488 0.6886 0.6946

0.9036 0.9139

0 −0.0948

0.528 0.7678 0.7738

0.9828 0.9931

0.0792 −0.0156

0.215 0.2193

102.4 409.6

0.6961

0.9174

−0.1544

0.7753

0.9966

−0.0752

0.2213

204.8

Figure 2. Cyclic voltammograms of the oxidation of 10−3 M solutions of β-carotene (a), 8′-apo-β-carotene-8′-al (b), and canthaxanthin (c) at a Pt disk electrode in CH2Cl2 (left) versus THF (right) with 0.08 M TBAHFP, scan rate 100 mV/s. Adapted from ref 23.

Table 2. Redox Potentials of Other Carotenoids Standardized to the Ferrocene/Ferrocenium Potential of 0.528 V vs SCE carotenoid

E10

E20

E30



ΔE


kcar/kst15

β-carotene canthaxanthin2,(4) 8′-apo-β-carotene-8′-al4 ethyl-8′-apo-β-caroten-8′-oate4 7′-apo-7′,7′-dicyano-β-carotene4

(0.54) (0.689) 0.72 0.722 0.739

(0.545) (0.894) 0.865 0.875 0.916

(0.035) (0.264) 0.075 0.205 0.238

0.634 (0.626) 0.775 (0.775) 0.795 0.797 0.825

0.605 (0.631) 0.972 (0.980) 0.94 0.95 1.002

−0.029 (0.005) 0.197 (0.205) 0.145 0.153 0.177

(0.121) (0.350) 0.15 0.28 0.324

0.5 2 3 12.5 24

2,(4)

al.2 The corrected values for astaxanthin, monoester, and diester listed in Table 1 are 0.7678, 0.7738, and 0.7753 V for E10 and 0.9828, 0.9931, and 0.9966 V for E20. The difference in oxidation potential ΔE = E20 − E10 for astaxanthin, monoester, and diester found to be 0.215, 0.219, and 0.221 V, respectively, is also listed in Table 1. The reduction potentials E30 (Table 1) for the formation of neutral radicals have values close to zero for all three studied carotenoids and appear as a low intensity peak 5 for all three carotenoids. Difficulty of Obtaining Reversible CVs. Reversible CVs are ideally needed to obtain accurate redox potentials. Previously published studies have demonstrated the difficultly of obtaining reversible CVs for carotenoids. Some asymmetric carotenoids are irreversible due to the less stable nature of their radical cations and dications. Once the symmetry of the carotenoid is altered, an irreversible CV occurs in which the cathodic peaks are very small, indicating that dications and radical cations decay faster at room temperature. For example, asymmetric 8′-apo-β-carotene-8′-al (see Figure 2, on the left),

ethyl-8′-apo-β-caroten-8′-oate,4,21 and 7′-apo-7′,7′-dicyano-βcarotene4 (carotenoids listed in Table 2) all have the cathodic peak 3 (which corresponds to the reduction of Car2+ to Car•+) greatly decreased, indicating the radical cations are unstable and fewer species Car2+ were generated in peak 2 from radical cations. The asymmetry and increasing the withdrawing effect of substituents causes radical cations and dications to be less stable. When the conjugated length factor is added,4,21 carotenoids containing an ester, aldehyde, or cyano group display irreversible cyclic voltammograms in which peaks 2, 3, and 4 are absent, showing that the stability of radical cations and dications decreases even more. Symmetric carotenoids like β-carotene, canthaxanthin (see Figure 2, on the left), or astaxanthin display a more reversible behavior, except for the fifth peak. One drawback in displaying a reversible behavior is dealing with the multitude of reactions listed in Scheme 1, which requires working in a dry atmosphere and in the absence of air. Besides this, other factors like the type of aprotic solvent (see 2334



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Figure 2 comparing CH2Cl2 and THF), type of electrolyte,22 and preparation of the working electrode influence this behavior. All carotenoids are soluble, and their radical cations have the longest lifetime in methylene chloride which can be easily maintained under anhydrous conditions as compared to other organic solvents.4 Other solvents than anhydrous methylene chloride need to be rigorously dried by established methods including triple vacuum distillation over metals. For example, when HPLC grade THF bubbled with nitrogen for 30 min and used without further purification was used,23 irreversible CVs were obtained (Figure 2, on the right). It was also shown22 that dications of carotenoids are more stable in 0.1 M TBAHFP than in the presence of the same concentration of TBAPC or TBATFB, making TBAHFP the best choice in working with carotenoids. For carotenoids listed in Table 2, all measurements were performed using anhydrous methylene chloride and 0.1 M TBAHFP, and with a very similar electrode setup. However, the measurement conditions were different from those in the current study, the fifth reduction peak due to formation of neutral radicals being prominent in the previous CV measurements.2,4,5 The absence of the peak for astaxanthin and its esters in this current study (see Figures 3−5) indicates the

Figure 5. Cyclic voltammograms of the oxidation of 0.0004 M solution of astaxanthin n-octanoic monoester in CH2Cl2 and using 0.1 M TBAHFP as supporting electrolyte.

distorts the CVs of carotenoids in previous studies was considered a result of rather annoying side reactions that would complicate simulation, and it was tried, without success, to be entirely gotten rid of. Dications which are strong acids (pKa ≈ −2) and radical cations which are weak acids (pKa = 4− 7) tend to lose a proton in the presence of water and form cations and neutral radicals, respectively (see eqs 5 and 6 in Scheme 1). Cations, upon reduction, also form neutral radicals according to eq 3 in Scheme 1. Deprotonation of the radical cation, along with that of the dication, was found to be a very important step in the mechanism used to simulate the experimental CV.5 In the experiments described here, anhydrous methylene chloride extracted from a Sure seal 99.99% bottle from Aldrich by an oven-dried glass syringe was used. No plastic syringes can be used, as the solvents will extract contaminants from the plastic. The absence of air is required, as reaction between radical cations and oxygen over the minutes of time needed to do electrochemical measurements generates peroxide radicals, an irreversible reaction that confuses the study. All glassware must be dried overnight in an oven at 120 °C to extract the surface water that adheres to the SiO surface of the glassware. It is important to have all items ready to do the electrochemical measurements in the shortest time practical. Carrying out the entire measurement in a drybox provided to be impractical, but with care and speed, it could be done on the benchtop. In this experiment, all glassware along with the working and counter electrodes were cleaned and rinsed with acetone, held in an oven overnight, and then transferred to the drybox. The cell, except for the reference calomel electrode, was assembled inside the drybox, and the carotenoid solution, prepared also inside a drybox, was added to it. The holes for the reference electrode and nitrogen stream were covered before taking the cell outside the drybox, and they were added rapidly to the cell outside. After running the first CV of astaxanthin without success, the working electrode was polished for 3 min and rinsed with acetone and then with methylene chloride. The hole left from removing the working electrode was covered for the entire duration of the polishing process. The surface of the working electrode must be carefully prepared by vigorous polishing before measurements. For example, in an extreme case of carotenoid adsorption on electrodes, a reported study showed that a 5400 MW conducting polymer grew on the end of the electrode, resulting in irreversible CVs with an intense reduction peak and making determination of accurate oxidation

Figure 3. Cyclic voltammogram of the oxidation of 0.0004 M solution of astaxanthin in CH2Cl2 and using 0.1 M TBAHFP as supporting electrolyte.

Figure 4. Cyclic voltammograms of the oxidation of 0.0004 M solution of astaxanthin n-octanoic diester in CH2Cl2 and using 0.1 M TBAHFP as supporting electrolyte.

absence or minimal presence of water in the system. The greatly reduced fifth peak for the three carotenoids makes the cyclic voltammograms nearly reversible. The fifth peak that 2335



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potentials impossible.24−26 The intense reduction peak was due to adsorption of species involved in generation of dications on the surface of electrodes.4 The CVs of astaxanthin were run in the first 12 min after polishing the electrode. A soft steam of nitrogen was passed through solution, making sure that the solution would not get agitated or start to evaporate. After recording astaxanthin, the covered monoester solution was taken from the drybox and the cells quickly switched outside after the electrodes have been thoroughly rinsed with methylene chloride. The monoester CVs were recorded in the first 8 min after assembling the cell. The diester CVs were also taken in the first 10 min after switching cells and rinsing the electrodes with methylene chloride. At the end of the experiment, a few drops of water were added to the diester solution to cause formation of neutral radicals and the appearance of the fifth peak. A ferrocene test was carried out which established a reference point (0.4488 vs SCE) so that redox potentials in different experiments can be compared. However, the potential of the ferrocene/ferrocenium redox couple has not been reported in most of the electrochemical studies of carotenoids. Furthermore, up until 2006, there were not reported values for redox potentials of astaxanthin except the first oxidation potential reported by Han et al.27 in methylene chloride versus Ag/AgCl. Despite the fact that the solvent was purified by passing through an alumina column, the CV shows decomposition. The reverse sweep lacks the reversible nature, and there is a considerable amount of the neutral radicals formed. Also, there is no reported calibration vs ferrocene. A second study of astaxanthin reported in 2010 by Polyakov et al.28 shows CVs that are reversible except for the fifth peak due to neutral radical formation. The first and second oxidation potentials in methylene chloride versus SCE were reported as 0.755 and 0.974 V; however, there was no calibration versus ferrocene performed. Comparison with Other Carotenoids. Table 2 lists redox potentials that were previously measured against ferrocene for symmetric and asymmetric carotenoids with different substituents. For comparison, they were standardized to the ferrocene/ferrocenium potential value of 0.528 V vs SCE given in ref 2. The first oxidation potential increases from β-carotene (E10 = 0.634 V), to canthaxanthin (E10 = 0.775 V), to astaxanthin and its esters (from Table 1, E10 = 0.7678, 0.7738, and 0.7753V), 8′-apo-β-carotene-8′-al (E10 = 0.795 V), 8′-apoβ-carotene-8′-oate (E10 = 0.797 V), and 7′-apo-7′,7′-dicyano-βcarotene (E10 = 0.825 V). The difference in oxidation potentials for the formation of the radical cation and the dication (ΔE) in Table 2 increases up to approximately 0.2 V when substituents are varied from electron donors like cyclohexene in β-carotene to electron acceptors like aldehyde in 8′-apo-β-carotene-8′-al, keto in 8′-apo-β-carotene-8′-oateand canthaxanthin, or dicyano in 7′-apo-7′,7′-dicyano-β-carotene. Substitution of two keto groups at positions C4 and C4′ of β-carotene leads to an increase of ΔE by 0.205 V in canthaxanthin. Addition of hydroxyl groups at positions C3 and C3′ of canthaxanthin which helps in extending the conjugation length like in astaxanthin, monoester, and diester causes further increase in ΔE to 0.215, 0.219, and 0.221 V, respectively (see Table 1). The slight difference in ΔE indicates that the ester group (for monoester or diester) has no essential influence on the oxidation potential because they do not essentially contribute to the length of the conjugated π system. Note that ΔE is smaller by approximately 0.050 V for asymmetric 8′-apo-β-

carotene-8′-al, 8′-apo-β-carotene-8′-oate, and 7′-apo-7′,7′dicyano-β-carotene (ΔE = 0.145, 0.153, and 0.177 V, respectively). The larger the ΔE, the larger is the comproportionation equilibrium constant Kcom for eq 4 in Scheme 1, and the greater is the ability to form radical cations in solution. The large equilibrium constants Kcom in Table 2 for astaxanthin, monoester, and diester, 102.4, 409.6, and 204.8, respectively, indicate that the monoester and diester would form radical cations slightly more easily than astaxanthin itself. On the basis of the fact that deprotonation of radical cations leads to formation of neutral radicals, all three carotenoids could be very efficient quenchers of excited singlet and triplet states29−32 with the esters of astaxanthin being probably better quenchers than astaxanthin. It is known15 that astaxanthin coordinates with metal ions to form metal complexes. The stability constant K1 when astaxanthin coordinates with a metal ion at one end is much larger than K2 when it coordinates at both ends; thus, the monoester would also coordinate with metal ions like astaxanthin. A diester will not be able to coordinate with metals; however, the large Kcom indicates that it also forms radical cations and thus neutral radicals available for quenching excited states. Importance of Carotenoid Neutral Radicals. Astaxanthin is a photoprotective pigment that can quench triplet-state Chl.33 With accumulation of astaxanthin in Haematococcus pluvialis, non-photochemical quenching of chlorophyll fluorescence was shown to increase drastically.34 It was suggested35 that the efficient radical trapping by astaxanthin at the surface and inside the phospholipid membrane is due to the orientation of the carotenoid in the membrane such that its two polar terminal rings are located at both polar surfaces of the lipid bilayer and its hydrophobic polyene chain is extended through the hydrophobic region of the membrane. Astaxanthin radical cations in an environment like that described above would readily give protons from the terminal rings to the polar media forming neutral radicals. A carotenoid neutral radical would be long-lived and very efficient in fluorescence quenching by J exchange for either an excited singlet or triplet state, analogous to fluorescence quenching by a stable nitroxide neutral radical situated as far away as 9 Å from a fluorescing molecule.36 An energetically favorable charge transfer state between the carotenoid radical cation and chlorophyll radical anion needs to occur, and upon formation of the radical cation, proton transfer to neighboring surroundings would form the neutral radical. The carotenoid should also be in close proximity to an excited Chl molecule for quenching to occur. DFT Calculations Prediction for Proton Loss. It is known that astaxanthin traps radicals not only at the polyene chain but also at the terminal rings, with the C3 methine position being suggested to be a radical trapping site.44 Deprotonation from the radical cation and dication to form neutral radicals according to eqs 3, 5, and 6 was investigated by DFT calculations. There have been numerous studies30,31,37,38 discussing deprotonation from the radical cations of carotenoids, including a recent study38 that investigated proton loss from the radical cations of astaxanthin, monoester, and diester. In the case of the radical cation, proton loss occurs from positions on the cyclohexene rings that extend the π-conjugated system. The most stable astaxanthin neutral radical forms by proton loss at the C3 (or C3′) position of the terminal ring followed by proton loss at the C5 (or C5′) methyl group and then by loss from the methyl groups of the polyene chain. For the diester, loss is prevented at the cyclohexene ends and is 2336



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noids with low oxidation potentials like β-carotene and zeaxanthin (∼0.60 V vs SCE) are oxidized by the ferric ion, and more radicals are generated in a Fenton reaction, known as the prooxidant effect. This effect was highlighted for zeaxanthin in a recent publication42 on photochemical and optical properties of xanthophylls. Astaxanthin is a “pure antioxidant”;43 it does not show any pro-oxidant effect.44 Astaxanthin and its esters are expected to show an antioxidant effect based on increasing oxidation potentials (0.7678, 0.7738, and 0.7753V, respectively). Furthermore, astaxanthin has been shown to coordinate with metal ions; this strong complexation could remove Fe3+ as an oxidant to produce more radicals generated (bad situation in bio systems) by Fenton chemistry. The stronger the electron accepting ability, the greater is the proton acidity of the donating proton, which is reflected in a greater oxidation potential. This dependence is also related to the length of the conjugated chain. The fewer the conjugated double bonds, the higher the oxidation potential. For example, CV measurements of aldehydes and esters21,45 with decreasing conjugation length and of carotenoids containing a triple bond that shortens the conjugation length4 show increased oxidation potentials. From spin trapping studies with a decrease in oxidation potential, there is a tendency to form radicals during a Fenton reaction.15 This is reflected in a lower kcar/kst ratio (Table 2). For example, β-carotene with a ratio 0.65 gets oxidized easily and more radicals are formed, while canthaxanthin, with kcar/kst = 2, is not oxidized as easily as βcarotene, and has a greater ability to react with •OOH by proton abstraction. The extremely high antioxidant activity of astaxanthin is consistent with a high oxidation potential (0.7678 V), while the monoester and diester (0.7738 and 0.7753 V, respectively) would also have similar scavenging abilities, respectively. The higher oxidation potentials of astaxanthin and monoester and diester make Fenton chemistry less likely, as these carotenoids scavenge radicals according to ref 15. Furthermore, in the presence of a base (like OH− in the Fenton reaction), the proton from the hydroxyl group which is the most acidic would come off, leaving a negative charge. Once the negative charge is formed, the proton from position C3 (C3′) cannot be lost (two negative charges next to each other). Thus, for astaxanthin, a proton would be abstracted from only two positions, the C5 and C5′ methyl positions. On the other hand, the monoester would have three positions available for trapping: C5′ at one end and positions C3 and C5 at the ester end. For the diester, with no hydroxyl groups attached, proton loss would probably occur at positions C3, C5 and C3′, C5′ at both ends; thus, four positions would be available for scavenging radicals. This might account for a small change in the scavenging ability of astaxanthin, monoester, and diester.

favored for its methyl groups. The monoester allows formation of the neutral radical at the C3′ position and prevents its formation at the opposite end with the ester group attached. Migration of a proton from the hydroxyl group to the carbonyl group facilitates resonance stabilization at the primed end of the monoester, and at both ends of astaxanthin.38 Our current DFT study at the B3LYP/6-31G** level determines the most stable astaxanthin cations #Car+ (n), where n indicates the position of proton loss, formed by proton loss from astaxanthin dication Car2+, as seen in Table 3 (also Table 3. Relative Energies (kcal/mol) from DFT Calculations (B3LYP/6-31G** Level) for Cations #Car+ (n) Formed by Proton Loss from Carotenoid Dication Car2+ According to eq 5 in Scheme 1; n Indicates the Position of Proton Loss Car #

Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+ # Car+

(15) (14) (13) (12) (11) (10) (9) (8) (7) (5) (4) (3) (3OH) (2) (1)

astaxanthin

β-carotene

46.4 44.4 36.0 35.9 52.8 38.1 29.9 33.0 55.33 22.7

31.4 29.5 21.3 26.8 35.7 23.7 15.5 19.1 37.5 5.1 0 47.8

0 30.7 34.9 27.5

45.1 14.9

compared to β-carotene) and Table S1 (in the Supporting Information, energies given in Hartrees). According to this study, the most favorable proton loss from astaxanthin dication also occurs from positions located on cyclohexene rings, rather than from the polyene chain. Proton loss from the astaxanthin dication is most favored at the C3 (or C3′) methine position, followed by proton loss at the C5 (or C5′) methyl group at a difference of 22.7 kcal/mol, proton loss from the C1 (or C1′) methyl groups (not investigated for proton loss from the radical cation) at a difference of 27.5 kcal/mol, and last from the methyl groups of the polyene chain. For β-carotene, which has protons available at methylene position C4 (or C4′), loss is most probable from this position rather than from the C3(or C3′) position. Loss is then favorable at the C5 (or C5′) methyl group by a 5.1 kcal/mol difference. Radical Trapping Ability. Besides showing the strongest singlet oxygen quenching activity among polyphenols, tocopherols, carotenoids, ascorbic acid, coenzyme Q10, αlipoic acid, and other hydrophilic and lipophilic antioxidants,39 astaxanthin has a powerful antioxidant activity toward free radicals like hydroxyl40 or peroxyl.41 It was shown15 by spin trapping experiments that the ability to react with •OH, •CH3, or •OOH radicals by proton abstraction from carotenoid is nonlinearly related to the first oxidation potential of the carotenoid. The plot was generated for carotenoids with varying oxidation potentials, natural and synthesized. Astaxanthin is known to have had some of the highest oxidation potentials among the natural occurring carotenoids. Carote-

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CONCLUSION Oxidation potentials of astaxanthin and n-octanoic monoester and n-octanoic diester of astaxanthin have been measured and calibrated versus ferrocene. To date, there were no ferrocenecalibrated potential measurements for astaxanthin. The potentials were compared to those of other carotenoids that were ferrocene-calibrated. Carefully calibrated potentials standardized to the ferrocene potential of Hapiot et al. show the behavior of β-carotene, canthaxanthin, 8′-apo-β-carotene-8′-al, ethyl-8′-apo-β-caroten-8′-oate, 7′-apo-7′,7′-dicyano-β-carotene, and astaxanthin and its n-octanoic acid monoester and diester as scavenging agents toward •OH, •CH3, or •OOH radicals. It is predicted that the n-octanoic acid astaxanthin monoester and 2337



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(10) Lotocka, M.; Styczynska-Jurewicz, E. Astaxanthin, Canthaxanthin and Astaxanthin Esters in the Copepod Acartia Bif ilosa (Copepoda, Calanoida) during Ontogenetic Development. Oceanologia 2001, 487−497. (11) Mordi, R. C. Carotenoids-Function and Degradation. Chem. Ind. 1993, 110, 79−83. (12) Woodall, A. A.; Lee, S. W.; Weesie, R. J.; Jackson, M. J.; Britton, G. Oxidation of Carotenoids by Free Radicals: Relationship Between Structure and Reactivity. Biochim. Biophys. Acta 1997, 1336, 33−42. (13) Mortensen, A.; Skibsted, L. H.; Sampson, J.; Rice-Evans, C.; Everett, S. A. Comparative Mechanisms and Rates of Free Radical Scavenging by Carotenoid Antioxidants. FEBS Lett. 1997, 418, 91−97. (14) Edge, R.; McGarvey, D. J.; Truscott, T. G. The Carotenoids as Anti-oxidants-A Review. J. Photochem. Photobiol., B 1997, 41, 189− 200R. (15) Polyakov, N. E.; Leshina, T. V.; Konovalova, T. A.; Kispert, L. D. Carotenoids as Scavengers of Free Radicals in a Fenton Reaction: Antioxidants or Pro-Oxidants? Free Radical Biol. Med. 2001, 31, 398− 404. (16) Fukami, H.; Namikawa, K.; Tomimori, N.; Sumida, M.; Katano, K.; Nakao, M. Chemical Synthesis of Astaxanthin N-Octanoic Acid Monoester and Diester and Evaluation of Their Oral Absorbability. J. Oleo Sci. 2006, 55, 653−656. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, revision D.01; Gaussian, Inc.: Wallingford, CT, 2009. (18) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (19) Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the ColleSalvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785−789. (20) Gao, Y. L.; Focsan, A. L.; Kispert, L. D.; Dixon, D. A. Density Functional Theory Study of the β-Carotene Radical Cation and Deprotonated Radicals. J. Phys. Chem. B 2006, 110, 24750−24756. (21) Deng, Y. Carotenoid Radical Cations and Dications Studied by Electrochemical, Optical and Flow Injection Analysis: Lifetime, Extended Chain Conjugation, and Isomerization Properties. Ph.D. Dissertation, The University of Alabama, Tuscaloosa, AL, 1999. (22) He, Z.; Kispert, L. D. Effect of Electrolytes and Temperature on Dications and Radical Cations of Carotenoids: Electrochemical, Optical Absorption, and High-Performance Liquid Chromatography Studies. J. Phys. Chem. B 1999, 103, 10524−10531. (23) Grant, J. L.; Kramer, V. J.; Ding, R.; Kispert, L. D. Carotenoid Cation Radicals: Electrochemical, Optical, and EPR Study. J. Am. Chem. Soc. 1988, 110, 2151−2157. (24) Gao, G.; Wurm, D. B.; Kim, Y.-T.; Kispert, L. D. Electrochemical Quartz Crystal Microbalance, Voltammetry, Spectroelectrochemical, and Microscopic Studies of Adsorption Behavior for (7E,7′Z)-Diphenyl-7,7′-Diapocarotene Electrochemical Oxidation Product. J. Phys. Chem. B 1997, 101, 2038−2045. (25) Gao, G.; Jeevarajan, J. A.; Kispert, L. D. Cyclic Voltammetry and Spectroelectrochemical Studies of Cation Radical and Dication Adsorption Behavior for 7,7′-Diphenyl-7,7′-Diapocarotene. J. Electroanal. Chem. 1996, 411, 51−56. (26) Gao, G. Electrochemical, Optical, Photochemical and Chromatographic Studies of Carotenoid Cation Radicals and Dications: Adsorption, Polymerization and Isomerization Properties. Ph.D. Dissertation, The University of Alabama, Tuscaloosa, AL, 1997. (27) Han, R.-M.; Tian, Y.-X.; Wu, Y.-S.; Wang, P.; Ail, X.-C.; Zhang, J.-P.; Skibsted, L. H. Mechanism of Radical Cation Formation from the Excited States of Zeaxanthin and Astaxanthin in Chloroform. Photochem. Photobiol. 2006, 82, 538−546. (28) Polyakov, N. E.; Focsan, A. L.; Bowman, M. K.; Kispert, L. D. Free Radical Formation in Novel Carotenoid Metal Ion Complexes of Astaxanthin. J. Phys. Chem. B 2010, 114, 16968−16977. (29) Hideg, E.; Barta, C.; Kalai, T.; Vass, I.; Hideg, K.; Asada, K. Detection of Singlet Oxygen and Superoxide with Fluorescent Sensors

n-octanoic acid diester would have similar abilities for proton abstraction by low molecular weight radicals like •OH, •CH3, and •OOH when compared to astaxanthin itself. According to DFT calculations, proton abstraction would occur from positions situated on the cyclohexene rings. The large oxidation potentials for the three carotenoids are consistent with the electron accepting nature of their substituents. The stronger the electron accepting ability of the terminal substituents, the higher the oxidation potential is and the more stable the radical cations are in dichloromethane. Large Kcom for astaxanthin and its esters indicates a preference for formation of radical cations; thus, neutral radical generation from these species is probable in conditions that allow deprotonation. Neutral radicals would be very efficient quenchers of excited singlet and triplet states.

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ASSOCIATED CONTENT

S Supporting Information *

Energies (in Hartrees) from DFT calculations (B3LYP/631G** level) of proton loss from carotenoid dication Car2+ to form cations #Car+ according to eq 5 in Scheme 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Faculty Research Seed Grants (FRSG) Program at Valdosta State University and in part by The Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Sciences, U.S. Department of Energy, grant DEFG02-86ER-13465 (L.D.K.), and by the Natural Science Foundation for EPR instrument grants CHE-0342921 and CHE-0079498.

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REFERENCES

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